J. Nannoplankton Res. 36 (1), 2016, pp.65–75 © 2016 International Nannoplankton Association
ISSN 1210-8049 Printed by The Sheridan Press, USA

65

Quantification of a pretreatment procedure for organic-rich
calcareous nannofossil samples
Jamie L. Shamrock*ExxonMobil Upstream Research Company, 22777 Springwoods Village Parkway, Spring, TX, USA 77389; *jamie.l.shamrock@exxonmobil.com

Jean M. Self-Trail
U.S. Geological Survey, 926A National Center, 12201 Sunrise Valley Dr., Reston, VA, USA 20192; jstrail@usgs.gov
Manuscript received 15th June, 2015; revised manuscript accepted 7th March, 2016

Abstract A new sodium hypochlorite (NaClO) pretreatment technique for calcareous nannofossil samples richin marine and terrestrial organic components was tested on samples containing moderate amounts of organic matter(~1–3%). In the untreated control slides, organic matter at moderate levels obscured individual specimens or caused
cohesion of nannofossils with background material, such as clays and opaque minerals. While samples with >7%
background organic matter require two iterations of the pretreatment procedure (Shamrock et al., 2015), samples
with average background values up to 3% were sufficiently treated with one exposure to the primary oxidizing
reagent (NaClO). Pretreated samples showed notable increases in both relative abundance and species richness. In
addition, we examined the potential loss of nannofossil specimens while decanting the supernatant after centrifuging.
Despite some negligible loss in nannofossil specimens, the enriched abundance and diversity of the data justifies thisenhanced processing procedure.

1. Introduction
Interest in unconventional resource exploration in the petroleum industry, as well as both academic and industrial
interest in carbon excursions, has resulted in a subsequent
increase in the number of nannofossil samples analyzed
from organic-rich lithologies. These samples are significantly more difficult to analyze than calcareous chalks andoozes due to the high proportion of marine and terrestrialorganics, clays and opaque minerals. Specimens are frequently obscured by organic matter and may adhere tolarger clumps of background material, which can lead toskewed abundance counts and reduced species richness.
To minimize these issues, a sodium hypochlorite pretreatment method for organic-rich samples was developed withexcellent results (Shamrock et al., 2015). Pretreated samples show significant reduction in residual organic matter as well as increases in both the number of identifiable
specimens and in species richness.
Samples from this original study had high (~7–14%)
values of µorg (average residual organic matter per field ofview [FOV]; N = 50 at 630x magnification), and requireat least two exposures to the oxidizing reagent (NaClO) to
produce significant results. However, samples with only
1–2% µorg can still significantly inhibit sample preparationand subsequent data collection. In the present paper, we
examine whether samples with a lower µorg can be significantly improved while reducing pretreatment to only one
exposure to the NaClO solution and documented the relative improvement in these lower µorg samples.
One potential pitfall of the procedure is the loss ofnannofossil specimens when decanting after centrifuging
the treated samples. Decanted specimens would likely bevery small in size, and several of these very small species

are used as marker taxa. In addition, changes in the relative abundance of some small species can act as significant
bioevents (e.g., acme events) or be important in paleoenvironmental analysis. As this step is repeated several times
throughout the pretreatment, the residual sediment in the
supernatant liquid was examined with respect to bothdried weight and relative floral composition of the residues, to quantify any negative impact this step may haveon nannofossil assemblage compositions.
2. Localities and samples
A total of twelve samples were examined for calcareous
nannofossil content. Seven samples were collected from thelate Cenomanian Clubhouse Formation and an unnamed
Turonian formation from the Holland Ball Park core, located in the City of Suffolk, VA (Figure 1; 36.68211°N,
76.78058°W). One sample was collected from the Eocene
Exmore Formation of the Ashby Subdivision core, located in
Carrollton, VA (Figure 1; 36.93317°N, 76.52973°W). Threesamples were collected from the late Campanian Donoho
Creek Formation in southern North Carolina (Figure 2)
from two outcrops on the Cape Fear River: Donoho Creek
Landing (34.48065°N, 78.40995°W), and Brown’s Landing (34.47505°N, 78.39767°W). One sample was collectedfrom the early Maastrichtian Peedee Formation at Mitchell’s
Landing (34.3567°N, 78.20947°W). Sample depths andsample codes are detailed in Table 1.
Cored samples were extracted from the central portionof freshly broken core in order to avoid contamination bydrilling fluid. Outcrop samples were collected by digging
back into the exposed sediments on the outcrops to obtainfresh, unweathered material. Samples from the Cape Fear
River, which is tidally controlled near the river mouth, are66

Shamrock, Self-Trailcentrifuge tube with 3ml NaClO reagent, a ~5–6% sodium
hypochlorite solution as found in basic household bleach.
Samples were shaken then submerged in a 100°C hot water bath for the prescribed time, then centrifuged using
a Thermo Scientific CL2 centrifuge with a 16.5cm rotor
diameter. The supernatant liquid was decanted, and samples were then washed with a 9.5pH bicarbonate solution
to neutralize any residual acidity within the sample fromthe break-down of organic matter. Finally, samples wererinsed several times with distilled water (See Table 2). It
is critical to rinse the bicarbonate ions out of the treatedsample, otherwise these residual ions will crystalize on the
coverslip, obscuring much of the final preparation.

Figure 1: Map showing the location of the Holland Ball Park core (HC)and the Ashby Subdivision core (AC). Note that the Ashby core is locatedin the outer fracture zone of the Chesapeake Bay impact crater. Modified
from Edwards & Powars (2003). Central crater outline updated according
to Powars et al. (2009)

recorded as “Taped Feet”. These samples are measured
upward from a permanent spike drilled into the base ofeach section, with the location of each spike recorded by
GPS and tied to base sea level.
3. Methodology and procedures
A complete and detailed description of the pretreatment
procedure and background methodology utilized for thisstudy is documented in Shamrock et al. (2015). Details
on procedure times, volumes, etc. are provided in Table 2.
Samples for this study were processed by first placing a small amount of sample (~0.1g) in a 15ml Kimax

3.1 Residual organic material andtreatment duration
All samples were initially prepared both as untreatedsmear slides using the double slurry method of Watkins &Bergen (2003) and using the pretreatment method above.
Samples were examined in PPL (plane polarized light)
on a Zeiss AxioImager A.2 microscope to assess µorg.
Relative abundance estimates of µorg were made using
Compton (1962) for abundances of 2–50% per FOV and
Reid (1985) for abundances ≤1% per FOV. A select set ofsamples with the highest µorg were reprocessed with only
one exposure to the NaClO solution to assess oxidation
potential in samples with lower residual organic material
than those studied in Shamrock et al. (2015). In addition,
we reexamined some samples with high nannofossil
abundance to assess potential effects of pretreatment on
nannofossil assemblages.
The pretreatment method was originally developed
using samples which all contained significant amounts ofmarine and terrestrial µorg (≈7–14%). A single exposure
to the sodium hypochlorite solution was insufficient forsuch organic-rich samples, and optimal results (µorg < 1%)were achieved with two exposures to the NaClO solution

Figure 2: Map showing the location of Donoho Creek Landing, Brown’s Landing and Mitchell’s Landing on the Cape Fear River. Stars indicate thelevel at which a sample was taken. Modified from Self-Trail et al. (2012)

Quantification of a pretreatment procedure for organic-rich calcareous nannofossil samples

SampleLocality

Depth (ft)Depth (m)Sample CodeFormation

67

Latitude (N)Longitude (W)36.68211

76.78058

36.93317

76.52973

34.48065

78.40995

Holland Core (VA)
1

Holland Core

273.8

83.45

HC 273.8

unnnamed Turonian2

Holland Core

317.0–317.2

96.62–96.68

HC 317.2

Clubhouse

3

Holland Core

337.4

102.84

HC 337.4

Clubhouse

4

Holland Core

347.2

105.83

HC 347.2

Clubhouse

5

Holland Core

369.0

112.47

HC 369.0

Clubhouse

6

Holland Core

376.2–376.3

114.67–114.70

HC 376.3

Clubhouse

7

Holland Core

397.0–397.2

121.01–121.07

HC 397.2

Clubhouse

332.7

101.41

AC 332.7

ExmoreAshby Core (VA)
8

Ashby Core
Cape Fear River (NC)9Donoho Creek Landing

18.0

5.49

CFR-DCL 18.0

Donoho Creek

10

Donoho Creek Landing

35.0

10.67

CFR-DCL 35.0

Donoho Creek

11

Brown Landing

23.0

7.01

CFR-BL 23.0

Donoho Creek

34.47505

78.39767

12

Mitchell's Landing

6.0

1.83

CFR-ML 6.0

Peedee34.35670

78.20947

Reagent

VolumeVortexH2O Bath

Centrifuge
Speed

Centrifuge
Time

(ml)

(sec)

(min)

(rpm)

(min)

1

Sodium hypocholrite(NaClO)

3

3–5

15

2000

7

1–2*

2

Bicarbonate solution(NaHCO3)

5

3–5

15

2500

10

2

3

Distilled H2O

5

5–10

NA

2000

10

3

Distilled H2O

5Iterations

Step

Table 1: Sample list detailing sample locality, sample depth (in ft and m), sample code (used throughout text and figures), geologic formation andlatitude/longitude. VA = Virginia, NC = North Carolina, USAof sediment was collected from the centrifuge tube on the
tip of a flat-headed toothpick, and evenly distributed overthe coverslip using the double-slurry method. Even sediment distribution was fairly easy to achieve, as the repeated mixing of the sample and washing with distilledwater during pretreatment is very effective in deflocculating clays as well as individual nannofossil specimens.

3.3 Sample loss
The loss of sample while decanting was examined with
Table 2: Summary of reagents, volumes, mixing times, heating durations,
respect to the weight of the dried residue in the supercentrifuge parameters and number of iterations. The sodium hypochlorite
natant liquid, the number of nannofossil specimens, thesolution is 5–6% NaClO by volume, as found in basic household bleach.
The bicarbonate solution (NaHCO3) is mixed to 9.5pH. *The number of species richness and the average size (length) of the specoxidation steps is dependent on average residual organic matter of the imens observed in smear slides.
The supernatant from Steps 1 and 2 cannot be colsample. Two iterations are required when µorg is ≥7% per FOV. Only one†
lecteddue to the presence of either sodium hypochlorite
exposure to the reagent is required when µorg is ≤3% per FOV. For the
final water wash, samples are swirled on low for a minimum of four hours. or bicarbonate in the solution (Shamrock et al., 2015). The
For a detailed methodology and procedure, see Shamrock et al. (2015)supernatant liquid from the three water washes of Step 3(Table 2) were collected in a 50ml beaker. The liquid was(Shamrock et al., 2015). In comparison, samples in this allowed to sit for 48hrs, which exceeds the required setstudy contained notably less µorg, typically between 1–3%, tling rates detailed by Geisen et al. (1999), to permit all
residual suspended material to settle to the bottom. The
thus we reexamined the potential for reducing treatment.
liquid was gently pipetted off, and the residues dried inan oven at 100°C for two hours. The dried residues were
3.2 Nannofossil abundance and species
weighed and then prepared as smear slides to assess therichness
Nannofossil samples were examined in XPL (cross-­ composition of the sediment (Table 4).
polarized light) and PPL at 1000x magnification. Species
richness and relative abundance counts were made by 4. Results
identifying all specimens across a full traverse on a 40mm 4.1 Residual organic material andcoverslip, or approximately 200 FOVs. Two additional treatment duration
traverses were made to scan for rare taxa, which were Several untreated smear slides showed relatively low regiven a value of one. Specimens covered by organic mate- sidual organic matter (µorg ≈ 1%); however, six samples
rial or those contained in sediment clumps were identified yielded significant amounts of µorg (>1.5%) to warrant
when possible. Obscured specimens were listed as uniden- further testing (Table 3). Though relatively low in µorg,
tifiable. Relative abundance is significantly affected by the sample AC 332.7 was also included due to the high nanamount of material used in sample preparation, so care nofossil abundance and clay content.
Those samples with the highest µorg were processed
was taken to ensure similar sediment thicknesses on allwith
only one exposure to the NaClO solution to examine
sample preparations. After pretreatment, a similar amount4>4 hrs†

NA

2000

10

1

68

Shamrock, Self-Trailthe potential for reducing pretreatment time. All samples
showed significant reduction in residual organic matter
after only one pretreatment (Table 3). These results are
best seen in photomicrographs in plane polarized light.
Plate 1 shows sample CFR-BL 23.0 from all three preparations in both XPL and PPL. Residual organic matter andclumping of nannofossil specimens was seen in the untreated smear slides (Plate 1–1). Preparations treated with
one and two exposures to the NaClO solution (Plate 1–2
and 1–3, respectively) showed a significant reduction inresidual organic matter. In addition, sediment clumps weredisaggregated and a significant amount of clay-sized particles had been removed. These factors helped to improve
sample preparation and aid in data analysis.
Two exposures to the NaClO solution are necessary
for samples with ≥7% µorg (See Shamrock et al., 2015:
Figure 1, Table 1, 2); however, the most organic-rich sample examined here (CFR-BL 23.0; µorg = 3.2%) achieved
optimum results (µorg < 1%) with only one exposure to
the oxidizing reagent (Table 3, Plate 1–2), indicatingthat samples with µorg ≈ 3% or less could be sufficiently
treated with only one oxidization step. There was no data
for samples with µorg ≈ 4–6% in either study and, as a result, we were unable to define the value where one would
need to increase from one to two iterations.
4.2 Nannofossil abundance and species
richness
Samples were examined for relative nannofossil abundanceand species richness as described above. As in the original
study, there was a decrease in the number of ‘unidentifiable’
specimens in treated samples (Table 3). This term refers to

0.1

5

0

5

5

NA

0.1

46

1

45

22

HC 337.4

2.6

0

0

0

0

0.1

0

0

0

0

0.1

0

0

0

0

HC 347.2

2.5

0

0

0

0

0.1

0

0

0

0

0.1

0

0

0

0

HC 369.0

2.3

7

3

4

3

0.1

19

2

17

10

0.1

17

3

14

11

HC 376.3

2.4

63

8

55

18

0.0

76

1

75

20

0.1

81

0

81

21

HC 397.2

1.3

62

3

59

21

0.1

71

0

71

23

AC 332.7

0.3

895

23

872

70

0.1

1177

3

1174

93

CFR-DCL 18.0

0.7

1349

25

1324

78

NA

0.0

1959

4

1955

96

CFR-DCL 35.0

1.0

410

46

364

56

NA

0.1

3073

0

3073

99

CFR-BL 23.0

3.2

1297

106

1191

75

0.1

1996

0

1996

97

0.1

2063

3

2060

90

CFR-ML 6.0

1.7

687

57

630

70

0.1

2824

1

2823

93

0.1

2628

1

2627

96

µorg per FOVµorg per FOV

NA
0.1

Sd

±0.02

1129

2

1127

89

Sd

Species Richness

UnidentifiableNA

21

Identifiable

Total Specimens

2

33

Species Richness22

Identifiable2

35

Unidentifiable4

1.1

Total Specimens

1.0

HC 317.2

Species Richness

Undentifiable

HC 273.8

Identifiable

Total Specimens

Pretreatment 2xµorg per FOV

Pretreatment 1x

Sample CodeSmear Slide

specimens obscured by organic matter or aggregated into
clumps with other background components, such as clays,organics and opaque minerals. The pretreatment process
lightened organic matter, removed a portion of clay-sizedparticles (µlength ≈ 2µm) and dissociated sediment aggregates, thus freeing individual specimens for identificationand more robust relative abundance counts.
All fossiliferous samples from the late CenomanianTuronian age Holland core displayed an increase in relative abundance and species richness with pretreatment(Table 3). As expected, the sample with the highest residual organic matter (HC 376.3, µorg = 2.3%) showed a notable increase in species richness, though not as dramatic as
the AC and CFR localities.
Sample AC 332.7 (late Eocene) is taken from the Exmore Formation, the sedimentary resurge material thatfilled the Chesapeake Bay impact crater following a bolide
impact ~35.0Mya (Edwards et al., 2009) (Figure 1). The
sample contained a relatively rich, well-preserved Priabonian assemblage, intermixed with reworked specimensof Late Cretaceous, early Eocene (Ypresian) and late Eocene (Bartonian) age. Both reworked and in situ assemblages showed good preservation with the pretreatment, as
well as increased relative abundance and species richness(Table 3). The observed increases in relative abundancewith pretreatment, in this and the original study, had been
primarily attributed to changes in background organic matter; however, µorg was very low in the untreated AC 332.7
smear slide (µorg = 0.3% per FOV). Examination of thesupernatant liquid from this sample showed a significant
fraction of clay-sized particles (~2–3µm) (Plate 2–3). This,accordingly, played a significant role in the enrichment±0.03

Table 3: Summary of average residual organic matter, relative nannofossil abundance and species richness data with varying sample preparations.
Organic matter estimates (µorg /FOV) were made at 630x magnification in plane polarized light (PPL) where N = 50 FOV. Estimates were madeusing Compton (1962) for abundances of 2–50% per FOV and Reid (1985) for abundances of 1% or less per FOV. “Unidentifiable” refers only
to specimens obscured by organic material, clay minerals or clumping of background sediments. Samples treated once show increases in both
relative abundance and species richness, linked to lightening of organic matter, dissociation of sediment aggregates and removal of clays during
sample processing. Sd = standard deviation

Quantification of a pretreatment procedure for organic-rich calcareous nannofossil samples

Lmin-maxµlength

(µm)

(µm)

Untreated Smear
Slide

Pretreated
Slide

69

Supernatant
Residue

Sample CodeSampleWeight(g)

Residue
Weight(g)% Loss

RA

SR

RA

SR

RA

SR

HC 376.3

0.136

0.004

2.9

3.4

3.4

63

18

81

21

1

1

HC 397.2

0.125

0.000

0.0

NA

NA

62

21

73

23

0

0

AC 332.7

0.108

0.000

0.0

1.6–5.6

2.6

872

70

1176

93

28

6

CFR-DCL 18.0

0.117

0.003

2.6

2.0–6.1

3.2

1324

78

1955

95

25

11

CFR-DCL 35.0

0.126

0.001

0.8

1.9–3.6

2.8

364

56

3073

99

13

5

CFR-BL 23.0

0.128

0.006

4.7

1.5–4.9

2.8

1191

75

2060

90

18

5

CFR-ML 6.0

0.129

0.002

1.6

1.7–4.8

2.9

630

70

2627

96

26

9

Nanno Specimens

Table 4: Data comparison from decanted supernatant residues. Residue weights are a minor portion of the original sample weight, and are comprised
primarily of clay-sized (1–3µm) non-nannofossil background components. Maximum, minimum and average length is provided for residue nannofossil specimens. Three rightmost columns show relative abundance (RA) and species richness (SR) of selected untreated smear slides, pretreatedsamples, and pretreated sample residues. Nannofossil specimens observed in supernatant residues show low nannofossil abundance and species
richness relative to the parent samplesof the >3µm sediment fraction and relative abundance ofsmaller calcareous nannofossils (Table 4). This is further
discussed in the Sample Loss section, below.
Samples from the Cape Fear River outcrops showedvery abundant, diverse and relatively well-preserved assemblages of Late Cretaceous (Campanian) age. The notably high species richness was a result of a well-developed
oligotrophic assemblage combined with a well-preserved
shelf assemblage. All pretreated samples showed a decrease in the number of unidentifiable specimens and an increase in relative abundance and species richness (Table 3).
Photomicrographs of untreated smear slides and pretreatedslides of sample CFR-ML 6.0 again showed a decrease inresidual organic matter, clay-sized particles and clumped
sediments (Plate 3). Comparable results were obtainedwith either one or two exposures to the oxidizing reagent(Table 3).
4.3 Sample loss
While our results with the pretreatment method are predominantly positive, the increase in sample processing
also provides the opportunity to negatively impact the fossil assemblage. A potential for specimen loss occurs while
decanting the supernatant liquid after centrifuging. While
this step enriched the samples by removing significant portions of clay-sized particles (≈2–3µm), there are also somecoccoliths in the 2–3µm size range that are subject to loss.
Seven samples with the greatest abundance of nannofossil specimens were tested for this type of potential
specimen loss. The dried weights of sediment in the decanted liquid are provided in Table 4. Residues are produced from six decanting steps but could only be collectedfrom three of these steps, as described above. Therefore,
actual residue weights may be twice as high, or higher,
than the measured values. Nevertheless, the weight of decanted residues is still a very small proportion of original
sample weight (Table 4).
Smear slides of the residues showed the primary
components to be very small (2–3µm) carbonate and siliciclastic background particles (Plate 2–3, Plate 4). Still,some nannofossil specimens were identified in all of theresidues except the relatively low-abundance sample HC
397.2 (Table 4). Nannofossil specimens in these residues
represented a very small fraction of the total abundance
and were generally restricted to very small specimens ofparticular taxa. In addition, all species observed in theresidue slides were also identified in the pretreated slides,
indicating no impact on sample diversity. Table 4 compares relative abundance and species richness for smear
slides, pretreated samples and supernatant residues produced from the pretreatment process. Table 5 details the
distribution of specimens identified in the supernatant residues and compares these values to the pretreated samples
from which the residues were derived. Species identified
in residues were generally very abundant in the pretreatedsample, and represented only a fraction of the total species
richness of the sample (Tables 4, 5).
The lengths of all nannofossil specimens observed in
the residue slides were measured to understand the size
fractionation of specimens during the pretreatment. The
mean length (µlength) of all nannofossils specimens identified in the residue slides is 2.9µm (min. = 1.5µm, max. =6.1µm, N = 111). As expected, most specimens (74%)
were very small (length ≤3.0µm, ±0.2µm), and the majority (92%) were ≤4.0µm (±0.2µm) (Table 4). The largest
specimen identified was 6.1µm; however, this specimen is
one of the very few larger-sized outliers identified from allof the residue slides.
The abundance of individual taxa in the residues islinked to the relative abundance of those taxa in the pretreated sample, with the most abundant small taxa being
observed in the supernatant residue as well. For example,
we observed sample four specimens of Repagulum parvidentatum in the residue slide of sample CFR-BL 23.0,
while >250 were counted in the pretreated slide. Similarly,
two specimens of Eiffellithus gorkae were identified in theresidue of sample CFR-ML 6.0, while 163 were identified
in the pretreated slide. Abundance data for the residues
is compared to the abundance of these taxa in the pretreated slides in Table 5. Sample HC 376.3 is omitted from
­Table 5 as only one specimen of Watznaueria barnesiae
was observed in the residue slide.

Cruciplacolithus sp.

Reticulofenestra haqii

Reticulofenestra minuta

Reticulofenestra minutula

Pontosphaera sigmoidalis

Zeugrhabdotus erectus

P

1195

95

5

56

68

6

27

68

14

214

37

27

38

R

23

11

1

1

3

1

2

1

1

8

1

1

3

P

3073

99

183

196

77

322

56

R

13

5

3

2

1

6

1

Zeugrhabdotus trivectus

1

Tranolithus minimus

151

Retecapsa crenulata

57

19

Prediscosphaera stoveri

126

5Prediscosphaera spinosa

Coccolithus minimus

Zeugrhabdotus dipplogrammus

Repagulum parvidentatum

Prediscosphaera cretacea

105

1

Placozygus fibuliformis

0.98

3

1

Helicolithus trabeculatus

CRF-ML 6.0

15

6

Eiffellithus gorkae

0.72

93

28

Discorhabdus ignotus

CFR-BL 23.0

1176

Corollithion signum

0.42

Chiastozyugus synquadriperforatus

CFR-DCL 35.0

P
R

Biscutum constans

1.89

Species Richness

CFR-DCL 18.0

Angulofenestrellithus snyderi

2.33

Relative Abundance

AC 332.7

Slide Preparation% Relative to Parent Assemblage

Shamrock, Self-Trail

Sample Code

70

P

2060

90

53

4

160

286

84

29

R

15

6

2

1

1

4

6

1

P

2627

96

78

71

163

58

153

55

3

66

30

R

26

9

7

1

2

1

6

2

1

4

2

Table 5: Comparison of nannofossil assemblage data from pretreated samples and supernatant residues from centrifuging. Sample HC 376.3 is notincluded as only one specimen of W. barnesiae was observed. P = pretreated parent sample; R = sample residue. The relative abundance of nannofossil specimens in one full traverse of the residue slide is very low when compared to the pretreated parent sample (Column 2). Specimens tend tobe small taxa, with species such as R. parvidentatum and B. constans most frequently observed. Please refer to Nannotax (ina.tmsoc.org/Nannotax3)
for species concepts of taxa listed in the table

The Ashby core sample shows a very abundant anddiverse mixed Cretaceous and Eocene assemblage. This is
the first sample containing Cenozoic forms that has been
examined using the pretreatment method, and contains ahigh abundance of small reticulofenistrids (2–5µm). No
large taxa were documented in the Eocene residues and,as expected, the observed size distribution of small reticulofenestrids is similar to that of Cretaceous taxa such as
Biscutum constans, Repagulum parvidentatum and Zeugrhabdotus erectus (Table 5).
5. Summary and conclusions
The nannofossil pretreatment method of Shamrock et al.,(2015) was reexamined to determine its utility on samples
with considerably lower residual organic material than theoriginal study. Results indicated samples with as low as~1–3% µorg can be notably improved with sample pretreatment. The pretreatment process lightens organic matter,dissociates sediment aggregates and reduces the 1–2µm
fraction of background sediment. As a result, pretreatedsmear slides showed consistent increases in relative abundance and species richness. In contrast to samples with
>7% µorg that required two iterations of the NaClO treatment, samples with ~3% or less were sufficiently treatedby reducing the procedure to only one exposure to the oxidizing reagent.
Nannofossil specimen loss was observed during thedecanting process, but was negligible when compared tothe primary assemblage. Decanted specimens were small

to very small, with µlength ≈ 3µm. The relative abundanceof small taxa in the residue was related to the relative
abundance of the taxa in the sample. This was generally
a few percent, or less, of the total relative abundance observed in the treated samples, and does not appear to affect relative abundance schemes, general biostratigraphy
or paleoenvironmental analysis. In fact, it may be argued
that with the recovery of additional taxa and increases
in both abundance and species richness, the pretreatedsamples may give more accurate data with respect toboth biostratigraphy and paleoenvironmental interpretation. Results of both studies show nannofossil samples
with varying amounts of residual organic matter can besignificantly improved with pretreatment, and this procedure may both enhance paleoenvironmental interpretations and increase potential biostratigraphic resolution inorganic-rich lithologies.
Acknowledgements
The authors would like to thank Jim Holl (ExxonMobil) for permission and encouragement to work on the project, and Kurt
Johnston (ExxonMobil), Ellen Seefelt (USGS) and Laurel Bybell(USGS) for their feedback and suggestions. We would also liketo thank Jeremy Young (University College London) and Matt
Hampton (Network Stratigraphic Consulting Ltd.) for their reviews and critiques, which greatly improved the manuscript. Any
use of trade, firm, or product names is for descriptive purposes
only and does not imply endorsement by the U.S. Governmentor ExxonMobil.

Quantification of a pretreatment procedure for organic-rich calcareous nannofossil samples

References
Compton, R.R. 1962. Manual of Field Geology. Wiley, New
York: 378 pp.
Edwards, L.E. & Powars, D.S. 2003. Impact damage to dinocysts
from the Late Eocene Chesapeake Bay event. Palaios, 18(3):275–285.
Edwards, L.E., Powars, D.S., Gohn, G.S. & Dypvik, H. 2009.
Geologic columns for the ICDP-USGS Eyreville A and B
cores, Chesapeake Bay impact structure: Sediment breccias,
1096 to 444 m in depth. In: G.S. Gohn, C. Koeberl, K.G.
Miller & W.U. Reimold (Eds). The ICDP-USGS Deep Drilling Project in Chesapeake Bay Impact Structure: Resultsfrom the Eyreville Core Holes. Geological Society of America Special Paper, 458: 51–89.
Geisen, M., Bollmann, J., Herrle, J.O., Mutterlose, J. & Young,
J.R. 1999. Calibration of the random settling technique forcalculation of absolute abundances of calcareous nannoplankton. Micropaleontology, 45(4): 437–442.
Powars, D.S., Catchings, R.D., Goldman, M.R., Gohn, G.S.,­Horton, J.W., Jr., Rymer, M.J., Gandhok, G. & Edwards, L.E.
2009. High-resolution seismic reflection images across the
ICDP-USGS Eyreville deep drilling site, Chesapeake Bay
impact structure. In: G.S. Gohn, C. Koeberl, K.G. Miller &W.U. Reimold (Eds). The ICDP-USGS Deep Drilling Projectin the Chesapeake Bay Impact Structure: Results from theEyreville Core Holes. Geological Society of America Special
Paper, 458: 209–233.
Reid, J.C. 1985. Comparison chart for estimating volume percentages of constituents in rocks and concentrations in therange of 1.0 to 0.1 volume percent. American Mineralogist,
70: 1318–1319.
Self-Trail, J.M., Christopher, R.A., Prowell, D.S., Harris, W.B.,
Aleman Gonzalez, W.B. & Seefelt, E.S. 2012. Cretaceous
geology of the Cape Fear River (part B): From Walkers Bluffto Hood Creek Landing. Geological Society of America: Abstracts with Programs, 44(7): 444.
Shamrock, J.L., Muñoz, E. & Carter, J.H. 2015. An improved
sample preparation for calcareous nannofossils in organic-­
rich mudstones. Journal of Nannoplankton Research, 35(2):101–110.
Watkins, D.K. & Bergen, J.A. 2003. Late Albian adaptive radiation of the calcareous nannofossil genus Eiffellithus. Micropaleontology, 49: 231–252.
Young, J.R., Bown, P.R. & Lees, J.A. (Eds). Nannotax3 website.
International Nannoplankton Association. 24 April 2015.
URL: http://ina.tmsoc.org/Nannotax3.

71

72

Shamrock, Self-Trail

Plate 11
Plate
Sample CFR-BL 23.0
Photomicrographs taken at 630x for wider FOV; Selected specimens highlighted for comparison
10µm

1a

XPL

1b

PPL

Untreated slurry-smear slide; organic matter particularly evident in PPL
10µm

2a

XPL

2b

PPL

Pretreated with one exposure to NaClO solution
10µm

3a

XPL

3b

Pretreated with two exposures to NaClO solution

PPLQuantification of a pretreatment procedure for organic-rich calcareous nannofossil samples

73

Plate 22
Plate
Sample AC 332.7 - Photomicrographs taken at 630x for wider FOV; Selected specimens
highlighted for comparison. Sample shows low µorg, but is improved by processing throughdissociation of sediment clumps and removal of clay-sized background material (3a-b)
10µm

1a

XPL

1b

PPL

Untreated slurry-smear slide
10µm

2a

XPL

2b

PPL

Pretreated with one exposure to NaClO solution
10µm

3a

XPL

3b

Residue slide produced from decanted supernatant

PPL

74

Shamrock, Self-Trail

Plate 33
Plate
Sample CFR-ML 6.0
Photomicrographs taken at 630x for wider FOV; Selected specimens highlighted for comparison
10µm

1a

XPL

1b

PPL

Untreated slurry-smear slide; organic matter particularly evident in PPL
10µm

2a

XPL

2b

PPL

Pretreated with one exposure to NaClO solution
10µm

3a

XPL

3b

Pretreated with two exposures to NaClO solution

PPLQuantification of a pretreatment procedure for organic-rich calcareous nannofossil samples

75

Plate 44
Plate
Photomicrographs of supernatant residues, taken at 630x. Decanted residues consist primarily of clay-sized(2-3µm) particles reflecting dominant sediment mineralogy (1, 2: CaCO3, 3: SiO2), with rare small nannofossil
specimens, highlighted for comparison.
10µm

1a

XPL

1b

XPL

Sample CFR-BL 23.0
10µm

2a

XPL

2b

PPL

Sample CFR-ML 6.0
10µm

3a

XPL

3b

Sample HC 369.0

PPL